Neocaridina denticulata

Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp

Neocaridina denticulataY.W. Sin et al./General and Comparative Endocrinology xxx (2014) xxx–xxx

Environmental Physiology of Marine Organisms

Van-Tinh Nguyen201356732

Department: Biomedical Engineering/ Marine Biomedical Science Laboratory.

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The arm of this study

• In this study, the authors identify genes involved in the biosynthesisand degradation of sesquiterpernoids, and biosynthesis ofecdysteroids in the newly sequenced cherry shrimp Neocaridinadenticulata.

• This information will provide an understanding of the evolution ofhormonal systems across the Pancrustacea and Arthropoda as awhole.

Keywords: Crustacean, Ecdysteroid, Juvenile hormone, Methyl farnesoate, Insect, Arthropod

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Introduction

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SHELLFISH (MOLLUSCS AND CRUSTACEA) j Characteristics of the Groupshttp://taxo4254.wikispaces.com/Balanus+Amphitritehttp://en.wikipedia.org/wiki/Neocaridinahttp://animaldiversity.ummz.umich.edu/

Neocaridina

The Crustacea is conventionally divided into 6 classes, the Branchiopoda, Maxillopoda, Ostracoda, Remipedia, Cephatocarida and Malacostraca

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Nathan J. Kenny et al., Mar. Drugs 2014, 12

Neocaridina denticulata Life Cycle

They have chosen N. denticulata for further study as it has much potential to become a model for aquacultural,developmental, ecotoxicological, food safety, genetic, hormonal, physiological and reproductive studies in thelaboratory at pH of 6.5–8.0, and temperature is 22–24°C

a) The lifecycle of N. denticulatab) The appearance of gravid female compared to a female without eggs. The scale bar represents 5 mm on three adult

shrimp.c) Some of the colour forms available commercially. (i) Red patched; (ii) punctate red patterning; and (iii) blue.

Sesquiterpenoid pathway and Ecdysteroid biosymthesic pathway genes

• The steroidogenic CYPs are encoded by the Halloween genesphantom (CYP306A1), disembodied (CYP302A1), shadow (CYP315A1)and shade (CYP314A1)

• Each of the Halloween enzymes is believed to mediate one specificenzymatic step in the biosynthesis of 20-hydroxyecdysone (20E), asmutations in these genes result in low ecdysteroid levels andembryonic death.

• Spook (CYP307A1), is believed to mediate an uncharacterized step inthe biosynthesis of 20E, as its mutation will also result in low 20Eproduction.

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Materials and methods

• Gene identification in N. denticulata genome resources.

• Gene sequences were identified in the N. Denticulata genome using TBLASTNsearches.

• Phylogenetic analysis.

• The Bayesian phylogenetic tree showing inter-relationships of a variety ofmalacostracan crustacean species, including the position of N. denticulate.

• RNA isolation, RT-PCR, subcloning and sequencing.

• To check if contigs containing different exons of some genes could be merged.Primers for PCR amplification of identified target genes were designed based onexonic regions.

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Results

• Juvenoid hormone biosynthesis

• Juvenoid hormone degradation

• Juvenoid hormone regulation

• Ecdysteroid production

• Regulators of both juvenoid hormones and ecdysteroids

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Juvenoid hormone biosynthesis

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Juvenile hormone pathway overview (biosynthesis). Genesidentified in Neocaridina denticulataare shown in boxes withdarker coloration (i.e. JHAMT, JHBP, JHEH, JHE and JHEBP).

In insects, juvenile hormone acid

methyltransferase (JHAMT) plays a major role in

the biosynthesis of juvenile hormones (JHs).

The last two steps of JH biosynthesis differ

depending on the insect group.

In the Lepidoptera, epoxidation by an epoxidase

converts FA to JH III acid and its homologues,

which are subsequently methylated by JHAMT,

whereas in most other insects epoxidation follows

methylation of Farnesoic acid (FA) to Methyl

farnesote (MF) by JHAMT.

JHAMT has only been reported in the

crustacean water flea D. pulex to date, but the

discovery of JHAMT in N. denticulate.

Figure S1 Amino acid sequence identity for JHAMT

of N. denticulata and other arthropods.11/25/2014 9

Hence, JHAMT is more generally present across

crustaceans and may play an important role in thebiosynthesis of Methyl farnesote (MF) in crustaceans.

Figure S25 Phylogenetic tree of JHAMT amino acid sequences from N.

denticulata and other arthropods, based on the 50% majority rule tree from the

Bayesian analysis.

Amino acid sequence identity for JHAMT of

N. denticulata and other arthropods

Figure S3 Amino acid sequence identity for juvenilehormone (JH) binding protein (JHBP) of N. denticulata

and other arthropods, which has been proposed to bethe cytoplasmic receptor of JH in insects.

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Expression of JHAMT in N. denticulate embryos, juveniles and adults.Expression of JHAMT in embryonic, juvenile and adult N. denticulatasamples asnoted.

JHAMT in the sesquiterpenoid biosynthetic pathway of crustaceans and insects indicates the JH biosynthetic pathway found in

insects may have already been present in the Pancrustacean as shown.

The JHAMT gene in embryonic, juvenile and adult samples, showing at least weak transcription is ubiquitous across the body of

this species, albeit very weak in posterior regions of juveniles and adults.

This suggests that JHAMT may be active around the body.

Expression of JHAMT in N. denticulate embryos, juveniles and adults

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Juvenile hormone pathway overview

(degradation). Genes identified in Neocaridina

denticulata are shown in boxes with darker

coloration (i.e. JHAMT, JHBP, JHEH, JHE and

JHEBP).

Juvenoid hormone degradation

Figure S4 Amino acid sequence identity for JHEBP of N. denticulata and

other arthropods.11/25/2014 12

Figure S2 Amino acid sequence identity for JHE of N. denticulata and

other arthropods.

Figure S5 Amino acid sequence identity for JHEH of N. denticulata and other

arthropods.

Amino acid sequence identity for JHE/

JHEBP/JHEH

Figure S6 Amino acid sequence identity for type A allatostatin of N.

denticulata and other arthropods.11/25/2014 13

Juvenoid hormone regulation

Allatostatins (ASTs) are well known neuropeptides that inhibit

the biosynthesis of juvenile hormones (JH) by the corpora allata

(CA) in insects, and several allatostatin-like peptides were

identified in the N. denticulate genome.

In insects, there are three major types of ASTs: A-type, muscle-

inhibiting peptide (MIP) type, and C-type.

Figure S9 Amino acid sequence identity for allatostatin receptors of N. denticulata and

other arthropods.11/25/2014 14

Figure S8 Amino acid sequence identity for type C allatostatin of N. denticulata and

other arthropods.

Figure S7 Amino acid sequence identity for muscle-inhibiting peptide (MIP) of N.

denticulata and other arthropods.

The MIP identified in N. denticulate also possesses amino acid sequence with

tryptophan in the second and ninth positions.

Juvenoid hormone regulation

Ecdysteroid productionEcdysteroid synthesis

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The steroidogenic CYP enzymes mediating steps in the conversion of cholesterol to

ecdysone in the crustacean molting gland (Y-organ), and the conversion of ecdysone to

20-hydroxyecdysone (20E) in the peripheral tissues.

The terminal hydroxylations at carbons are catalyzed by enzymes encoded by Spook,

Phantom, Disembodied, Shadow, and Shade.

A central tenet of insect biology is that ecdysteroids mediate larval-larval molts in the

presence of the sesquiterpenoid, JH.

In the absence of JH, by contrast, ecdysteroid induces a larval-pupal transition.

Ecdysteroid 20E regulates insect development and reproduction.

Molting is triggered by ecdysteroid, which is secreted from a pair of Y-organs. The Y-

organ is normally degenerates after puberty, and no subsequent molts occur.

Teruaki Nakatsuji, et al., General and Comparative Endocrinology,135,3,2004,358-364

Transductory cascade of Prothoracicotropic Hormone (PTTH)

interaction with a prothoracic gland cell. S6*, multiple

phosphorylated form.

Halloween genes encode P450 enzymes that mediate steroid

hormone biosynthesis in Drosophila melanogaster

Halloween gene disembodied, plays a critical role in

ecdysteroidogenesis

Molecular and Cellular Endocrinology, Volume 215, Issues 1–2, 2004, 1 - 10

Abbreviated scheme of ecdysteroidogenesis.

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Ecdysteroid pathway genes

Figure S18 Amino acid sequence identity for spook(CYP307A1) of N. denticulata and other arthropods.

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Figure S19 Amino acid sequence identity for phantom(CYP306A1) of N. denticulata and other arthropods.

Amino acid sequence identity for

spook/phantom

Figure S20 Amino acid sequence identity for disembodied(CYP302A1) of N. denticulata and other arthropods.

11/25/2014 19Figure S21 Amino acid sequence identity for shadow(CYP315A1) of N. denticulata and other arthropods.

Amino acid sequence identity for

disembodied/shadow

Figure S22 Amino acid sequence identity for Cytochrome P450-18a1

(CYP18A1) of N. denticulata and other arthropods.20

Figure S24 Phylogenetic tree of CYP (cytochrome P450) amino acid sequences from N.

denticulata and other arthropods, based on the 50% majority rule tree from the Bayesian

analysis. The substitution model selected was Blosum, with 99.7% posterior probability.

Bayesian posterior probabilities above 50% are shown above the branches.

Amino acid sequence identity for Cytochrome

P450-8A1/ Cytochrome P450

Regulators of both juvenoid hormones and ecdysteroids

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Figure S10 Amino acid sequence identity for

Methoprene-tolerant (Met) of N. denticulata and

other arthropods.

Met can also interact with Ecdysone receptor

(EcR) and retinoid X receptor (RXR) in their

binding to ecdysone response elements, so 20E could

drive molting and metamorphosis.

The action of ecdysteroids is mediated through a

heterodimer of two members of the nuclear receptor

family, the EcR and Ultraspiracle (Usp).

The USP protein is an ortholog of the RXR.

Figure S11 Amino acid sequence identity for EcR of N. denticulata and other

arthropods.

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Figure S12 Amino acid sequence identity for RXR of N. denticulata and other arthropods.

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Figure S14 Amino acid sequence identity for Broad-complex of N.

denticulata and other arthropods.


Broad expression eventually disappears during the early pupal stage

coinciding with the onset of adult cuticular synthesis. The role of Broad

was further defined by applying sufficient JH to pupae to elicit the

formation of a second pupal cuticle.

Broad’s presence in the cell appears to inhibit larval cuticular

synthesis, while permitting pupal cuticular synthesis to proceed.

Figure S15 Amino acid sequence identity for Chd64 of N. denticulata and other arthropods.

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Chd64 and FKBP39 could mediate the action of MF-Met on the MF response element

Figure S16 Amino acid sequence identity for FKBP39 of N. denticulata and FKBP39, FKBP46

and FK506 of other arthropods.

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Figure S17 Amino acid

sequence identity for

CHH/MIH/GIH of N.

denticulata and other

arthropods. They are

functions for potential

regulator of MF.

Figure S23 Phylogenetic tree of Crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-

inhibiting hormone (CHH/MIH/GIH) amino acid sequences from N. denticulata and other arthropods.


Conclusions

• This study has been suggested that the complete juvenile hormone (JH) andecdysteroid pathways were present in the insect-crustacean common ancestor,and these regulatory systems could possibly also date back to the arthropodancestor.

• However, many questions remain, could the JH and ecdysteroid systems haveevolved concurrently with the emergence of the exoskeleton in arthropodradiation?

• How are these different pathways components regulated in vivo in differentarthropods?

• The investigations of their regulation and functions in different arthropods,including the crustaceans, would be of immense importance for research onaquaculture, development, endocrinology.

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Thank you for your attention

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